Highly active antiretroviral therapy (HAART) has markedly prolonged the survival of individuals infected with human immunodeficiency virus type 1 (HIV-1), but there exists an important need for intervention in the transmission of the virus itself. Indeed, there is a continual emergence of drug-resistant viruses, as well as a dramatic increase in the number of HIV-1 cases in resource-poor countries where the currently available therapies for HIV-1 infection are too expensive for wide spread use. Thus, there remains a need for the development of compositions and methods for the prophylactic treatment of HIV-1.
Current antiretroviral drugs inhibit the HIV-1 virus following its entry into cells. An important step forward in the fight against AIDS would be the availability of compositions capable of acting in the early events of virus replication, i.e., which would inhibit the fusion of the viral envelope with the plasma membrane of target cells. Drugs endowed with this characteristic activity are called “fusion inhibitors.” Such drugs are being extensively sought in the effort to inhibit HIV-1 replication. As the events leading to HIV fusion are mediated by the viral envelope glycoproteins, these glycoprotein molecules represent the principal target for fusion inhibitors.
Fusion of the viral envelope of HIV with target cells consists of four steps: (1) primary interaction of the HIV envelope glycoprotein (gp120) with the human CD4 surface antigen, which acts as the principal HIV receptor; (2) conformational changes in both gp120 and CD4; (3) binding of gp120 to members of the chemokine-receptor family (CXCR-4 for the X4 HIV strains and CCR5 for the R5 strains), which act as HIV co-receptors; and (4) fusion between the cellular and viral membranes mediated by the gp41 viral envelope glycoprotein.
One strategy for fusion inhibition is to use peptides which mimic portions of the enveloping glycoproteins crucial for the early events in HIV replication. Peptides mimicking portions of the gp41 glycoprotein of HIV have shown in vitro anti-HIV activity and are currently being studied in clinical trials. One of them (T20, enfurtiuide) has recently been approved for use as a new anti-HIV drug in humans.
Another important target in the HIV envelope glycoproteins is the V3 loop of gp120, which is a crucial determinant for virus infectivity. The best described function of the V3 loop is its involvement in gp120 binding to chemokine receptors, but there are also data documenting the binding of the V3 loop to cell surface glycosphingolipids (GSL's) and CD4. Hammache, D., et al., “Specific interaction of HIV-1 and HIV-2 surface envelope glycoproteins with monolayers of galactosylceramide and ganglioside GM3,” J Biol Chem, 273:7967-71 (1998); Benjouad, A., et al., “Multibranched peptide constructs derived from the V3 loop of envelope glycoprotein gp120 inhibit human immunodeficiency virus type 1 infection through interaction with CD4,” Virology, 206:457-64 (1995). Interactions involving the V3 loop are described in U.S. Pat. No. 5,622,933 to Sabatier entitled “Multiple branch peptide constructions for use against HIV”, which is incorporated herein in its entirety. The V3 loop appears to stabilize gp120 attachment to CD4 by binding, through its tip, the CDR3 region of the D1 domain of CD4, which is uninvolved in the primary interaction with gp120. Treatment strategies utilizing peptides from the V3 loop have been discouraging, however, due to the high degree of variation shown in the V3 sequences of the different HIV strains, and also by the high immunogenicity of these sequences.
The lack of available, effective vaccines has spawned interest in the topical administration of drug therapies, which would limit the sexual transmission of HIV, referred to herein as “topic microbiocides”. Topic microbiocides would either kill the virus or, if the topic microbiocide belongs to the fusion inhibitor family, block entry of the virus into the body (not strictly a microbicidal action, but the term as used herein encompasses the inhibition action). The only therapy that has been extensively used for this purpose is nonxynol-9. A major drawback of this therapy, however, is that it can cause inflammation in the vaginal mucosa, therefore increasing rather than limiting HIV transmission. Other therapies have been postulated for this use as well. However, most of them are also likely to cause inflammation as they are derived from foreign tissues or sources. For these reasons, there is a need for a topic microbicide that inhibits HIV transmission without causing inflammation or toxic side effects. Such a therapy would greatly increase the safety of people who do not use condoms during sex, it would enhance the safety profile for those who do use condoms, and it could be used by women as a method of self-protection. In the search of topical microbicides with these characteristics, lessons can be learned from human proteins that are endowed with anti-HIV-1 effects.
Among the many proteins that are in some way associated with HIV-1 infection, the CD38 antigen merits particular consideration for use as an HIV-1 inhibitor due to its peculiar characteristics. Human CD38 is a type II surface glycoprotein with a molecular weight of 45 kD. As described in Savarino et al., “Role of CD38 in HIV-1 infection: an epiphenomenon of T-cell activation or an active player in virus/host interactions?”, AIDS vol. 14, no. 9, 1079-89 (2000), the contents of which are hereby incorporated in their entirety, human CD38 is composed of a short intracytoplasmic tail, a single transmembrane region and a long extracellular domain. CD38 is thought to exert three functions on T cells: (1) as an ectoenzyme, it leads to the formation of cyclic ADP-ribose, a crucial compound in regulation of intracellular Ca.sup.2t; (2) as an adhesion molecule, it mediates the interactions between leukocytes and vascular endothelial cells; and (3) as a molecule involved in transmembrane signaling, its engagement costimulates cell activation. In lymphocytes, surface CD38 has a peculiar pattern of expression, being expressed at high levels by recent thymic emigrants (RTE's), lost during maturation and re-expressed upon lymphocyte activation. Among T cells, it is detectable at high levels on mature thymocytes and activated T cells and at low levels on resting (i.e., HLA-DR.sup.-CD25.sup.-CD69−) nave cells (CD45RA+/R0− cells), whereas it is undetectable on resting memory cells (CD45RA−/R0+ cells).
The association between CD38 expression and lymphocyte activation makes CD38 a useful marker of progression in HIV disease, where generalized lymphocyte activation accompanies the progression of AIDS. Indeed, activation-related increases in CD38 molecules on both CD8+ and CD4+ T cells predict disease progression in HIV-1-infected adults. Conversely, decreased CD38 expression in both CD8+ and CD4+ T-cell subsets is a marker of effective response to HAART.
The available evidence indicates that there must be more to CD38 expression than its presence as an epiphenomenon of lymphocyte activation in HIV-1 infection. Indeed, CD38 is capable of interactions with some of the surface molecules involved in HIV-1 infection. The best documented of these interactions is the lateral association with CD4, the main HIV-1 receptor. Moreover, the CD38 molecule has been shown to be capable of interacting with cell surface GSL's. GSL's are organized in functional microdomains that have been compared to rafts moving on the plasma membrane. These rafts are associated with specific membrane proteins such as CD4. Recent studies suggest that GSL may also participate in HIV-1 fusion to CD4+ cells. According to these studies, the GSL microdomain may help stabilize the attachment of the virus to CD4 through multiple low affinity interactions between the V3 domain of gp120 and the carbohydrate moiety of GSL. Interestingly, data developed by the present inventors and by others indicates that CD38 is preferentially expressed in membrane rafts.
Finally, the CD38 molecule may affect lymphocyte susceptibility to HIV-1 infection. Studies by the present inventors showed that CD38 expression was negatively correlated to susceptibility to HIV-1 infection in human lymphoid cell lines, and that transfection of CD38 into CD38-cells conferred partial resistance to replication of both laboratory-adapted HIV-1 strains and primary isolates. Savarino, A., et al., “Investigation of the potential role of membrane CD38 in protection against cell death induced by HIV-1,” J Biol Regul Homeost Agents, 10:13-18 (1996); Savarino, A., et al., “Effects of the human CD38 glycoprotein on the early stages of the HIV-1 replication cycle”, The FASEB Journal, 13:2265-2276, 1999. These observations are in line with in vivo studies of others reporting that most HIV-1 infected cells are CD38− in the early stages of infection in vivo, although the activated (CD38+) portion of infected cells produce the highest levels of HIV-1 RNA. Zhang, et al., “Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells”, Science, 286:1353-1357 (1999). Based on this combined evidence, the present inventors recently published a model wherein HIV-1 gene expression displayed by some activated T cells would be due to post-entry events. Savarino, et al., “Role of CD38 in HIV-1 infection: an epiphenomenon of T-cell activation or an active player in virus/host interactions?”, AIDS vol. 14, no. 9, 1079-89, 2000.
The present inventors evaluated the specificity and generality of the anti-HIV-1 effects of CD38 and defined the critical domains of the protein involved in its inhibitory effects. The inventors show that, in transfected MT-2 cells, CD38 is expressed in rafts and renders these cells partially resistant to HIV-1 fusion. The present invention relates to the CD38 down-modulation of gp120 attachment to CD4. These effects are not shared with other molecules interacting with CD4. Thus, using truncated forms of CD38, the present inventors identified a sequence reminiscent of the V3 loop of gp120 in the extracellular portion. Peptides containing this sequence replicated the effects of CD38 and inhibited X4 and R5 primary isolates from different HIV-1 subtypes without detectable toxicity. Conversely, deletion of the first six amino acids in the V3-like sequence abrogated HIV-1 inhibition by CD38. Accordingly, the present inventors have identified peptide sequences from the CD38 leukocyte surface antigen which can inhibit or limit HIV transmission, or inhibit HIV replication, in vitro or in vivo.